The glycation of human serum albumin (HSA) during diabetes is believed to affect the transport, displacement, and non-bound plasma concentrations of drugs during this disease. HSA is a blood protein known to interact with a wide range of drugs. Glycation occurs when glucose undergoes a non-enzymatic reaction with amine groups on HSA to form stable fructosamine residues, which can then lead to other products. High-performance affinity chromatography (HPAC) using immobilized HSA in columns has been shown to be a fast and precise tool for studying the thermodynamics, kinetics, and stoichiometries of drug-protein interactions. HPAC is also valuable in studying drug-drug competition, identifying the binding regions for a drug on HSA, and measuring association constants for a drug at its various binding sites. We have recently shown that HPAC can be an effective and powerful method for examining drug interactions with glycated HSA. In this project, we will seek to continue obtaining such information by creating and employing highly novel HPAC assays and columns that contain or examine glycated HSA and exploring HPAC as a tool for examining interactions in heterogeneous protein populations and complex biological systems. Work in this project will be accomplished through three sets of studies that can be conducted in parallel. The first section will seek to develop novel entrapment-related techniques for HPAC to provide more rapid and detailed information on the interactions that occur between drugs and glycated HSA. These methods will be demonstrated and used in this project to examine the changes in binding by various drugs and in drug-drug interactions that occur upon the glycation of HSA. The second section will explore the creation of new HPAC methods that can provide a direct and rapid assessment of drug-protein binding in solution. These methods will be complementary to the more detailed binding studies using entrapped proteins, as conducted in the first section, by providing a means for screening solution-phase protein samples for binding to drugs and by providing a route that can be easily implemented for use with clinical samples. These methods will be used, in particular, to examine the variations in drug-HSA interactions that occur between and within diabetic patients. The third section will examine the creation and use of new affinity/mass spectrometry methods for simultaneously examining the structural and functional changes that occur in glycated HSA as related to drug interactions. A specific application to be examined for these methods will be to provide more detailed information on the relative role played by early vs. advanced glycation end-products in affecting drug interactions with glycated HSA. This research is relevant to public health because it will provide a better understanding of how diabetes affects the ability of HSA to bind drugs and other compounds. In addition, the HPAC methods and other tools developed in this project could be employed in the study of interactions in other heterogeneous systems, such as work with proteins that have undergone various degrees of glycosylation, phosphorylation, or alternative forms of post-translational modification.